Over 76,000 Americans will be diagnosed with melanoma this year alone and regrettably 10,000 of these patients will die of their disease. Oncogenic mutations in NRAS and BRAF genes are present in over 75% of melanoma. However, currently there is no effective treatment for NRAS mutant melanoma and targeted therapies against BRAF mutant melanoma fail to provide long-term clinical benefits due to rapid emergence of drug resistance. We postulated that identifying novel Druggable Genetic Vulnerability (DGV) pathways will lead to the development of alternative and effective therapies for NRAS mutant and targeted therapy resistant BRAF mutant melanoma. Towards this end, by employing an innovative approach of an in vivo druggable genome RNAi screen, we identified LIM-domain Kinase 2 ( LIMK2) as a novel drug target in melanoma. RNAi and CRISPR/CAS9-based inhibition of LIMK2 blocked the tumor and metastatic growth of NRAS- and BRAF-mutant melanoma and LIMK2 overexpression conferred resistance to BRAF- and MEK- targeted therapies. Our central hypothesis is that LIMK2 regulates a druggable genetic vulnerability pathway in NRAS/BRAF mutant melanoma, which is also important for conferring resistance to targeted therapies. The overall objective is to determine the role of LIMK2 in melanoma initiation and progression, understand its mechanism-of-action and evaluate LIMK2 as a drug target for melanoma therapy. In Aim 1, we will determine the role of LIMK2 in melanoma initiation and metastatic progression. Towards this end, we will first determine if LIMK2 is necessary for oncogenic NRAS/BRAF-induced transformation. Additionally, based on our results that LIMK2 promotes invasion and survival of circulating melanoma cells by conferring anoikis resistance, we will determine the in vivo role of LIMK2 in melanoma metastasis using xenograft- based organ-specific and spontaneous mouse model of melanoma metastasis. In Aim 2, we will determine the mechanism by which LIMK2 sustain tumor and metastatic growth of NRAS/BRAF mutant melanoma. Towards this end, based on the results of our unbiased high-throughput approaches of transcriptome-wide mRNA sequencing and Tandem Affinity Purification/Mass spectrometry analysis, we will evaluate the role of a novel LIMK2 substrate Dual Specificity Phosphatase 1 (DUSP1) as a potential downstream mediator of LIMK2 function in melanoma. In Aim 3, we will determine the role of LIMK2 in conferring resistance to BRAF- and MEK-targeted therapies and evaluate its utility as a drug target in melanoma. Towards this end, we will determine if LIMK2-driven DGV pathway inhibition prevents or delay emergence of resistance to BRAF- and MEK-targeted therapies and if systemic pharmacological inhibition of LIMK2 is effective in treating metastatic and BRAF- and MEK-targeted therapy resistant melanoma in vivo in xenograft-based and genetic mouse models of melanoma growth and metastasis. Taken together, our proposal will uncover a novel DGV pathway that can be pharmacologically targeted for treating metastatic and drug resistant melanoma.